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Allosteric control of ligand-binding affinity using engineered conformation-specific effector proteins

Nature Structural & Molecular Biology volume 18, pages 437–442 (2011)Cite this article

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Abstract

We describe a phage display methodology for engineering synthetic antigen binders (sABs) that recognize either the apo or the ligand-bound conformation of maltose-binding protein (MBP). sABs that preferentially recognize the maltose-bound form of MBP act as positive allosteric effectors by substantially increasing the affinity for maltose. A crystal structure of a sAB bound to the closed form of MBP reveals the basis for this allosteric effect. We show that sABs that recognize the bound form of MBP can rescue the function of a binding-deficient mutant by restoring its natural affinity for maltose. Furthermore, the sABs can enhance maltose binding in vivo, as they provide a growth advantage to bacteria under low-maltose conditions. The results demonstrate that structure-specific sABs can be engineered to dynamically control ligand-binding affinities by modulating the transition between different conformations.

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Figure 1: Phage display selection strategy.
Figure 2: Influence of sABs on maltose binding.
Figure 3: Scatchard analysis of maltose binding.
Figure 4: Crystal structure of MBP–MCS2 complex.
Figure 5: The 'wedge' formed by the CDR loops of MCS2.
Figure 6: Rescuing binding function of an MBP mutant.
Figure 7: Allosteric activity of sABs in vivo. Escherichia coli cells expressing sABs in the periplasm were grown in minimal media containing maltose as the sole carbon source.

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Acknowledgements

This work was supported by US National Institutes of Health grants GM 072688 and F32DK080619-02 (to S.S.R.). Use of the Advanced Photon Source was supported by the US Department of Energy, Office of Science, Office of Basic Energy Sciences, under contract no. DE-AC02-06CH11357. Use of the LS-CAT Sector 21 was supported by the Michigan Economic Development Corporation and the Michigan Technology Tri-Corridor (grant 085P1000817). The authors thank A. Koide (University of Chicago) for providing the phage library.

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  1. Department of Biochemistry and Molecular Biology and the Institute for Biophysical Dynamics, University of Chicago, Chicago, Illinois, USA

    Shahir S Rizk, Marcin Paduch, John H Heithaus, Erica M Duguid, Andrew Sandstrom & Anthony A Kossiakoff

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  1. Shahir S Rizk
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Contributions

S.S.R. and A.A.K. designed the experiments and wrote the manuscript; S.S.R., M.P., J.H.H., E.M.D. and A.S. performed the experiments; E.M.D. solved the crystal structure.

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Correspondence to Anthony A Kossiakoff.

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Rizk, S., Paduch, M., Heithaus, J. et al. Allosteric control of ligand-binding affinity using engineered conformation-specific effector proteins. Nat Struct Mol Biol 18, 437–442 (2011). https://doi.org/10.1038/nsmb.2002

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